Project Summary/Abstract Learning and memory are fundamental brain functions, yet their underlying cellular and neural circuit mechanisms remain poorly understood. Odor memories are exceptionally robust in humans and animals, of outstanding importance for survival and reproduction, and highly susceptible to neurodegenerative disorders including Alzheimer Disease. The olfactory (piriform) cortex, where odor perception is thought to first emerge, has long been suggested to encode odor memories. However, the cellular substrates and circuit mechanisms of olfactory learning are unknown. Our long-term goal is to understand the cellular and neural circuit mechanisms of odor perception and memory. The objective of this proposal is to provide a mechanistic cellular/molecular understanding of how odor memories are encoded and expressed. To achieve this objective we have developed activity-based intersectional genetic approaches in mice that allow us to identify and manipulate the activity of piriform neurons that were activated during olfactory learning. Our overall hypothesis is that odors activate sparse, distributed and functionally diverse piriform neurons, whose activity is necessary and sufficient for olfactory learning and memory. Aim 1: To determine how manipulating the activity of odor memory trace cells alters behavior. We will use genetic tagging based on cFos promoter activity (?Fos- tagging?) to visualize and manipulate the activity of piriform neurons that were activated during olfactory learning. Our preliminary data provide strong evidence for the necessity of Fos-tagged piriform ensembles for odor fear memory recall. Aim 2: To determine how learning alters the odor response properties of piriform ensembles. We will perform chronic two-photon imaging of odor-evoked activity in awake, behaving mice, before, during, and after aversive and appetitive olfactory conditioning. We will also selectively analyze the response properties of Fos-tagged piriform neurons that are essential for fear odor memory recall. We will test the hypothesis that olfactory learning selectively enhances the encoding of stimulus detection and discriminability in neurons constituting an olfactory memory trace. Aim 3: To determine the molecular identity and connectivity of olfactory memory trace cells. To drive behaviors, piriform ensembles must convey odor information to downstream target areas. Using our previously identified set of marker genes and single cell transcriptomics we will determine the molecular identities of piriform neurons that are activated during learning, and we will trace their axonal projections. We will test the hypothesis that olfactory learning facilitates functional connectivity of piriform cortex with task-relevant target areas. This project is innovative because is combines state-of-the-art genetic, behavioral, imaging and molecular approaches to identify the cellular and neural circuit substrates for olfactory learning and memory. It is significant because it will have a strong impact on the understanding of the neurobiology of memory loss in Alzheimer Disease patients.